In 1994 I was diagnosed with celiac disease, which led me to create Celiac.com in 1995. I created this site for a single purpose: To help as many people as possible with celiac disease get diagnosed so they can begin to live happy, healthy gluten-free lives. Celiac.com was the first site on the Internet dedicated solely to celiac disease. In 1998 I founded The Gluten-Free Mall, Your Special Diet Superstore!, and I am the co-author of the book Cereal Killers, and founder and publisher of Journal of Gluten Sensitivity.

By Scott Adams

Published on 07/26/1996

The following detailed explanation
of serological tests for celiac disease was written by Tom

The following detailed explanation
of serological tests for celiac disease was written by Tom Ryan,
Technical Service Specialist, INOVA
Diagnostics, Inc.

There has been a lot of discussion about serological testing for
celiac disease recently, specifically regarding tTG (tissue Transglutaminase)
testing. I will try to answer some of the many questions that have
appeared on this list about all of the tests. First, and this applies
to any of the blood tests, you must currently be on a gluten containing
diet for the tests to be accurate. Antibodies are produced by the
immune system in response to substances that the body perceives
as threatening. The immune response that your body produces is its
response to being exposed to gluten in the diet and its subsequent
effect on the intestinal mucosa. If there is no gluten in the diet,
then there is no response that we can measure. A brief change in
diet will not have a noticeable effect. If you have been gluten
free for a week or so, it will not make any great difference. The
response might be marginally less but the difference is insignificant
because the body has not had time to respond to the change. Conversely,
if you have been gluten free for a protracted period of time and
decide to be tested, a brief challenge of a couple of weeks is not
enough to elicit a response and get an accurate test.

There
are several steps that take place to generate an immune response
and it takes time both for the positive reaction when gluten is
present and to clear the antibodies when gluten is eliminated. There
has been a great deal of discussion about how much and how long
a challenge should be and there is no consensus. Talk with your
Doctor. My personal feeling is that the minimum is 2 slices of bread
per day for 6 weeks to get an accurate test but I would not try
to second-guess the Doctor. There are basically four tests that
can be performed to aid in diagnosing celiac disease. Notice that
I say they will aid in diagnosing celiac disease. Immunology
is fairly accurate but it is far from being an exact science. All
of the lab tests, regardless of the type or source, are presented
as aids to diagnosis. They should not be used alone as a basis for
diagnosis but rather are intended to be considered in conjunction
with the physical examination of the patient as well as the reported
symptoms, etc. by a trained physician. There has been a great deal
of confusion about what the tests are and I hope to alleviate some
of the misunderstandings. There are many terms that we hear. tTG,
IgA, IgG, ELISA, etc. What are all of these? Some contributors to
the list make reference to the IgA or IgG
test or to the ELISA test. These labels are incomplete
for our purposes and could be referring to any number of different
tests.

We
all have, within our bodies, a family of closely related although
not identical proteins which are capable of acting as antibodies.
These are collectively referred to as immunoglobulins.
Five major types of immunoglobulins are normally present in the
human adult. They are IgG, IgA, IgM, IgE and IgD. Each of these
is a shorthand way of writing immunoglobulin gamma G
(or A or M, etc.) and they each perform a different function in
our systems. IgG is the principal immunoglobulin in human serum.
It is important in providing immunity in a developing fetus because
it will pass across the placental barrier. IgA is the principal
immunoglobulin in secretions from respiratory and intestinal mucosa.
IgE is a gamma globulin produced by cells lining the intestinal
and respiratory tracts. It produces the antibodies associated with
most hypersensitivity (allergic) responses. It is associated with
asthma, hay fever, etc. IgM is a globulin formed in almost every
immune response in the early part of the reaction. IgD is a rare
protein present in normal sera in a tiny amount. These designations
refer to the type of protein that is carrying the antibody in question.
Both IgG and IgA subtypes of anti-gliadin antibody are produced,
hence we refer to them as IgG gliadin or IgA gliadin.
Collectively they are anti-gliadin antibodies.

Anti-Gliadin
Antibodies:

Both IgA and IgG anti-gliadin antibodies (AGA) are detected in sera
of patients with gluten sensitive enteropathy (celiac disease).
IgG anti-gliadin antibodies are more sensitive but are less specific
markers for disease compared with IgA class antibodies. IgA anti-gliadin
antibodies are less sensitive but are more specific. In clinical
trials, the IgA antibodies have a specificity of 97% but the sensitivity
is only 71%. That means that, if a patient is IgA positive, there
is a 97% probability that they have celiac disease. Conversely, if the patient
is IgA negative, there is only a 71% probability that the patient
is truly negative for celiac disease. Therefore, a positive result is a strong
indication that the patient has the disease but a negative result
does not necessarily mean that they don not have it. False positive
results are rather uncommon but false negative results can occur.
On the other hand, the IgG anti-gliadin antibodies are 91% specific
and have an 87% sensitivity. This means that they will show positive
results more readily but there is not as strong a correlation with
celiac disease. It is less specific. Patients with other conditions but not
afflicted with celiac disease will occasionally show positive results. IgG anti-gliadin
antibodies are detectable in approximately 21% of patients with
other gastrointestinal disorders. This test might yield false positive
results but is less likely to yield false negative results.

A
sensitive testing protocol includes testing for both IgA and IgG
anti-gliadin antibodies since a significant portion of celiac patients
(approx. 2-5%) are IgA deficient. This combined IgA and IgG anti-gliadin
antibody assay has an overall sensitivity of 95% with a specificity
of 90%. The type of test used to detect the anti-gliadin antibodies
is called an ELISA. This is an acronym and it stands for Enzyme
Linked Immuno-Sorbent Assay. ELISA is not a test in
itself. It is a method of testing and it is a relatively simple
test to perform. It involves putting a measured amount of diluted
patient serum into the wells of a specially constructed and prepared
plate and incubating it for a period of time with various chemicals.
The end result is a color change, the intensity of which is dependent
upon the concentration of anti-gliadin antibody (or other protein
being measured) in the patient serum. The ability of this colored
solution to absorb light at a particular wavelength can be measured
on a laboratory instrument and mathematically compared with solutions
that contain a known amount of anti-gliadin antibody to arrive at
a number for the amount of antibody present. The sample can then
be classified as negative, (0-20 units); weak positive, (21-30 units);
or moderate to strong positive if greater than 30 units. The purpose
of testing for anti-gliadin antibodies includes, in addition to
diagnosis of gluten sensitive enteropathy, monitoring for compliance
to a gluten free diet. IgA gliadin antibodies increase rapidly in
response to gluten in the diet and decrease rapidly when gluten
is absent from the diet. The IgA anti-gliadin antibodies can totally
disappear in 2-6 months on a gluten free diet, so they are useful
as a diet control. By contrast, IgG anti-gliadin antibodies need
a long time, sometimes more than a year, to become negative. The
reverse is also true. That is, a patient with celiac disease who has been on
a gluten free diet and tests negative for IgA anti-gliadin antibodies,
will show a rapid increase in antibody production when challenged
by gluten in the diet. Approximately 90% of challenged patients
will yield a positive IgA anti-gliadin result within 14-35 days
after being challenged. The IgG antibodies are somewhat slower.

Endomysial
Antibodies:

IgA
class anti-endomysial antibodies (AEA) are very specific, occurring
only in celiac disease and DH. These antibodies are found in approximately 80%
of patients with DH and in essentially 100% of patients with active
celiac disease. IgA endomysial antibodies are more sensitive and specific than
gliadin antibodies for diagnosis of celiac disease. Antibody titers (dilutions)
are found to parallel morphological changes in the jejunum and can
also be used to reflect compliance with gluten-free diets. Titers
decrease or become negative in patients on gluten free diets and
reappear upon gluten challenge.

The
test for anti-endomysial antibodies is more subjective and more
complicated for the lab to perform than the anti-gliadin assays.
It involves serially diluting some of the patients serum, that is,
diluting it by ½ then ¼, 1/8, 1/16, etc. and putting these dilutions
on a glass slide that has some sort of tissue affixed to it. The
slide is then processed with various solutions and examined under
a fluorescent microscope to determine if any of that serum binds
to any of the proteins in the tissue. If so, then that patient is
confirmed as having antibodies to that particular protein. This
method of testing is called an IFA or sometimes IIFA. It stands
for Indirect Immuno-Fluorescent Assay. The selection of which tissue
slide to use is determined by what specific protein, hence which
antibody, you are specifically looking for. Endomysial antibodies
react with the endomysium, which is a sheath of reticular fibrils
that surround each muscle fiber. Therefore, to detect endomysial
antibodies, you would want to use a tissue substrate that contains
a lot of muscle tissue. The substrate used most often for this assay
is distal sections of the esophagus. These are very thinly sliced
and fixed to the slide. They contain muscle fibers and not much
else so there is a lot of endomysium available to react with the
anti-endomysial antibodies.

Reading
this test involves viewing the reacted slides with a fluorescent
microscope to make the determination. This requires a highly skilled
and trained eye and, of necessity, is somewhat subjective. You are
looking for a green fluorescence in the endomysium covering the
muscle fibers. The test is reported as the titer or
final dilution in which the fluorescence can still clearly be seen.
As you can imagine, this is very subjective. There are no standardized
values and it is up to the judgment of the particular technician
what the endpoint titer is. Recently, (1998) the endomysial antigen
targeted by the anti-endomysial antibodies was identified as the
protein cross-linking enzyme known as tissue transglutaminase (tTG).
This has enabled the production of an antigen specific ELISA assay
incorporating tTG as a reliable and objective alternative to the
traditional and subjective Immunofluorescence based assays. In clinical
trials, the correlation with the endomysial IFA assay has been shown
to be close to 100%. This is a test that has been very well received
in the professional community. It is an ELISA, like the anti-gliadin
antibody test and, as such, is not subject to interpretation like
the IFA. That is the greatest advantage to this new test! With this
or any ELISA, the response is measured on an instrument that calculates
the amount of light of a particular wavelength that is absorbed
by the solution and prints out a numerical result. There is no chance
of human error skewing the results because there is no judgment
call involved. The ELISA plate, regardless of what you are testing
for, is processed with at least three control sera (sometimes as
many as eight) in addition to the unknown sample being tested. There
is a negative serum and at least two positive sera containing different
levels of the antibody being tested. There are specific requirements
for the absorption levels of these three controls. That is, each
of them has a minimum or maximum (or both) number that must be seen
by the instrument in order for it to be a valid test. If there is
any variance from these expected numbers, it is an indication that
something went wrong and the test results are discarded and the
test repeated. There is therefore no way the technician could report
inaccurate results, (assuming they diluted the sample correctly).
Either the test was valid, and you can rely upon the accuracy of
the result, or the test is invalid, and the entire result discarded.
If any error was made during the processing of the ELISA plate,
it would result in the control sera numbers being out of range and
the entire test result would be thrown out.

In
summary, the tTG ELISA is measuring the same thing that the endomysial
IFA is measuring but with a method that is more sensitive and specific
and not subject to interpretation. IgA class Reticulin antibodies
are found only in Celiac disease and dermatitis herpetiformis. These
antibodies are found in approximately 60% of celiac disease patients and 25%
of DH patients. This test is falling into disuse because of the
limited utility and the availability of better tests. It is an IFA
performed on a tissue substrate with all the attendant problems
that go along with it. The development of all of these serum assays
has tremendously simplified the diagnosis of celiac disease and improved the
accuracy as well. The original criteria for diagnosis according
to the European Society for Pediatric Gastroenterology and Nutrition,
(ESPGAN), involved a year of arduous studies with:

An initial
positive gut biopsy;

6 months
on a gluten free diet;

A second,
negative gut biopsy;

A gluten
challenge for 6 months and;

A
third, positive gut biopsy. The revised ESPGAN criteria call for
positive results in two of the serological tests confirmed by
a single positive biopsy. In practice, many gastroenterologists
are utilizing the serologies in conjunction with a controlled
diet and the clinical presentation to form a basis for diagnosis
without the need for the invasive procedure.

Through
the auspices of the Celiac Disease Foundation and others, a professional
symposium and workshop was organized earlier this year in Marina
Del Rey, California with participants from Europe as well as the
U.S. to establish standards for reporting test results. This should
improve testing and diagnosis even more. At the conclusion of this
conference a Celiac Disease Standardization Committee was formed
to investigate and make recommendations on a standardized method
of reporting results.